Clostridium difficile is a major nosocomial pathogen that causes severe diarrheal disease that is highly infectious and difficult to treat. C. difficileis easily transmitted due to the formation and expulsion of contagious spores from infected hosts. The spore form of C. difficile is resistant to most disinfectants and is critical for the survival f the bacterium outside of the host. The only natural environment known to support C. difficile spore formation is the gastrointestinal tract, yet we know very little about how spore formation occurs. We hypothesize that sporulation of C. difficile is initiated in response to signals found within the host intestine, through unknown and unique genetic mechanisms. The long-term goal of this project is to uncover the intestinal environment triggers C. difficile sporulation. The specific objectives of this application are to identify the genetic mechanisms required for spore initiation and determine how sporulation is initiated in the host. Capitalizing on our previous experience with Bacillus subtilis sporulation, Gram-positive intestinal pathogenesis and C. difficile molecular genetics, we will meet these objectives through the experiments detailed in two specific aims. First, we will reveal the genetic mechanisms of spore initiation in C. difficile through genetic analysis of sporulation-defective mutants and newly identified early sporulation regulators. In parallel, we will evaluate the effects of identified and suspected sporulation-inducing compounds on the timing and frequency of spore formation. The research proposed in this application is innovative because it represents a departure from the model sporulation paradigm as the archetypal standard for spore initiation by C. difficile and seeks to uncover the unique mechanisms that regulate sporulation in this pathogen. The expected contribution of the proposed research is a detailed understanding of the genes required for spore initiation and the development of spores within the host. This contribution is significant because it is the first ste in the development of strategies to prevent spore formation by C. difficile.